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Dual Specificity Phosphatases Support Axon Plasticity and Viability

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Abstract

In peripheral neuropathies, axonal degeneration (AxD) impairs the prognosis for recovery. Here, we describe a role for dual specificity phosphatases (DUSPs; MAP kinase phosphatases, MKPs), in supporting autonomous axon plasticity and viability. Both DUSPs 1 and 4 were identified within intact or axotomized sensory neurons. Knockdown of DUSP 1 or 4 independently or combined impaired neurite outgrowth in adult dissociated sensory neurons. Furthermore, adult sensory neurons with DUSP knockdown were rendered sensitive to axonopathy in vitro following exposure to low, subtoxic TrpV1 (transient receptor potential cation channel subfamily V member 1) activation by capsaicin, an intervention normally supportive of growth. This was not prevented by concurrent DLK (dual leucine zipper kinase) knockdown. Ex vivo neurofilament dissolution was heightened by DUSP inhibition within explanted nerves. In vivo DUSP knockdown or inhibition was associated with more rapid loss of motor axon excitability. The addition of SARM1 (sterile alpha and TIR motif containing 1) siRNA abrogated DUSP1 and 4 mediated loss of excitability. DUSP knockdown accelerated neurofilament breakdown and there was earlier morphological evidence of myelinated axon degeneration distal to axotomy. Taken together, the findings identify a key role for DUSPs in supporting axon plasticity and survival.

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Acknowledgments

Twinkle Joy provided technical assistance.

Funding

The work was supported by an operating grant from the Canadian Institutes of Health Research (FRN148675).

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Authors and Affiliations

  1. Neuroscience and Mental Health Institute and Division of Neurology, Department of Medicine, University of Alberta, 7-132A Clinical Sciences Building, 11350-83 Ave, Edmonton, AB, T6G 2G3, Canada

    Ambika Chandrasekhar, Prashanth Komirishetty, Aparna Areti, Anand Krishnan & Douglas W. Zochodne

  2. Department of Anatomy, Physiology, and Pharmacology, University of Saskatchewan, Saskatoon, Canada

    Anand Krishnan

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  1. Ambika Chandrasekhar
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  2. Prashanth Komirishetty
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  3. Aparna Areti
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  4. Anand Krishnan
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  5. Douglas W. Zochodne
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Contributions

AC participated in initial drafts of the paper, set up and designed experiments, helped to supervise the overall project, collated and analyzed results, and contributed to the intellectual content of the work. PK, AA, and AK set up and designed experiments for the work, collated and analyzed results, and contributed to the intellectual content of the work. DZ supervised the project, directed its overall methodology, and generated drafts of the paper.

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Correspondence to Douglas W. Zochodne.

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ESM 1

Supplemental Figure 1: (A,B) Longitudinal images of proximal stump of axotomized (3 days) mouse sciatic nerves colabelled with neurofilament (red) and either DUSP1 (green[A]) or DUSP4 green[B]) and DAPI (blue). The inset confirms axon double labeling (inset, yellow) for DUSP1 but not DUSP 4 (inset). The findings confirm DUSP1 axon localization in proximal stumps after axotomy whereas DUSP4 was expressed in periaxonal glial cells. Bar= 25 µM (PNG 6057 kb)

High resolution (TIF 21128 kb)

ESM 2

Supplemental Figure 2: (A,B) Longitudinal images of distal stumps of axotomized (3 days) mouse sciatic nerves colabelled with neurofilament (red in A, green in B) and either DUSP1 (green[A]) or DUSP4 (red [B]) and DAPI for nuclei (blue). The inset confirms axon double labeling (inset, yellow) for DUSP1 but not DUSP 4 (inset). The findings confirm DUSP1 axon localization in distal stumps after axotomy whereas DUSP4 was expressed in periaxonal glial cells. Bar= 25 µM (PNG 6628 kb)

High resolution (TIF 21127 kb)

ESM 3

Supplemental Figure 3: (A-C) DUSP1 siRNA knockdown had no impact on sensory neuron sprouting or branching. Comparable numbers of neurons were available for analysis in the scrambled compared to DUSP1 siRNA groups [n=5/group]. (D-F) DUSP4 siRNA knockdown was associated with reduced sprouting and branching. Comparable numbers of neurons were available for analysis in the scrambled compared to DUSP4 siRNA groups. Results from A-F are from the same experimental groups used to measure neurite outgrowth described in Figure 1. [#p=0.04, scrambled vs. DUSP4 siRNA, one tailed unpaired Student’s t-test, n=3/group; *p=0.0021, scrambled vs. DUSP4 siRNA, two tailed unpaired Student’s t-test, n=3/group] (PNG 360 kb)

High resolution (TIF 21124 kb)

ESM 4

Supplemental Figure 4: (A-C) DLK siRNA knockdown was associated with reduced sprouting (A) and reduced branching (B). Comparable numbers of neurons were available for analysis in the scrambled compared to DUSP4 siRNA groups (C). Results from A-C are from the same experimental groups used to measure neurite outgrowth described in Figure 5A-D. [#p=0.034 % sprouting scrambled vs. DLK siRNA, one tailed unpaired Student’s t-test, n=4/group; *p=0.051 branching scrambled vs. DLK siRNA, two tailed unpaired Student’s t-test, n=4/group] (PNG 410 kb)

High resolution (TIF 21125 kb)

ESM 5

Supplemental Figure 5: (A-C) DUSP 1,4 siRNA knockdown, with or without capsaicin, scrambled siRNA with a DUSP inhibitor with or without capsaicin and DLK siRNA with a DUSP inhibitor with or without capsaicin were associated with reduced sprouting (A) and reduced branching (B). DUSP 1,4siRNA knockdown, with capsaicin, scrambled siRNA with a DUSP inhibitor with or without capsaicin and DLK siRNA with a DUSP inhibitor with or without capsaicin were associated with lower numbers of harvested neurons available for analysis (C). Results from A-C are from the same experimental groups used to measure neurite outgrowth described in Figure 4,5. [*p=0.015 ANOVA for % sprouting; *p=0.0002 ANOVA for branching; *p=0.045 for numbers; n=3 for carrier inhibitor, 3 for carrier capsaicin and 4 for all other groups] (PNG 158 kb)

High resolution (TIF 21120 kb)

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Chandrasekhar, A., Komirishetty, P., Areti, A. et al. Dual Specificity Phosphatases Support Axon Plasticity and Viability. Mol Neurobiol 58, 391–407 (2021). https://doi.org/10.1007/s12035-020-02119-6

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